Guanylhydrazones useful for treating diseases associated with T cell activation

ABSTRACT

There is disclosed a method for treating diseases and disorders involving T cell activation and HIV-infection, using the p38 mitogen activated protein kinase (MAPK) signaling pathway as a target for intervention. There is further disclosed a use for guanylhydrazone-substituted compounds to treat diseases and disorders related to T cell activation and HIV-infection.

CROSS REFERENCE TO RELATED APPLICATION

This patent application is a division of application Ser. No.08/970,973, filed Nov. 14, 1997, now U.S. Pat. No. 6,143,728, issuedNov. 7, 2000, which claims priority from Provisional Patent ApplicationNo. 60/031,061, filed Nov. 15, 1996.

TECHNICAL FIELD OF THE INVENTION

The present invention provides a genus of guanylhydrazone-substitutedcompounds that are useful for treating various diseases associated withT cell activation and retroviral infection. The present invention isbased upon the mechanistic discovery that the p38 mitogen activatedprotein kinase (MAPK) signaling pathway is inhibited byguanylhydrazone-substituted compounds.

BACKGROUND OF THE INVENTION

The human immunodeficiency virus (HIV) has been implicated as theprimary cause of the slowly degenerative immune system disease termedacquired immune deficiency syndrome (AIDS) (Barre-Sinoussi et al.,Science 220:868-870, 1983; Gallo et al., Science 224:500-503, 1984). Inhumans, HIV replication occurs prominently in CD4⁺T lymphocytepopulations, and HIV-infection leads to depletion of this cell type andeventually to immune incompetence, opportunistic infections,neurological dysfunctions, neoplastic growth, and ultimately death. HIVis a member of the lentivirus family of retroviruses (Teich et al., RNATumor Viruses, 1984, Weiss et al., eds., CSH-Press, pp. 949-956). Otherretroviruses include, for example, oncogenic viruses such as human Tcell leukemia viruses (HTLV-I,-II,-III), and feline leukemia virus.

HIV-infection is pandemic and HIV-associated diseases represent a majorworld health problem. Although considerable effort is being put into thedesign of effective therapeutics, currently no curative anti-retroviraldrugs against AIDS exist. For example, virally encoded reversetranscriptase has been one focus of drug development. A number ofreverse-transcriptase-targeted drugs, including 2′,3′-dideoxynucleosideanalogs such as AZT ddI, ddC, 3TC, and d4T have been developed whichhave been shown to be active against HIV (Mitsuya et al., Science,249:1533-1544 (1990). While beneficial, these nucleoside analogs are notcurative (Larder et al., Science, 243:1731-1734 (1989). In addition, thedrugs often cause toxic side effects such as bone marrow suppression,vomiting, and liver function abnormalities.

The late stages of HIV replication, which involve crucial virus-specificprocessing of certain viral encoded proteins, have also been suggestedas possible anti-HIV drug targets. Late stage processing is dependent onthe activity of a viral protease, and drugs are marketed which inhibitthis protease (Erickson, Science 249:527-533, 1990). Thus, although agreat deal of effort is being directed to the design and testing ofanti-retroviral drugs, effective, non-toxic treatments are still needed.

Mitogen Activated Protein (MAP) kinases are important mediators ofsignal transduction from the cell surface to the nucleus. The ERK1 andERK2 mammalian subtype of the MAP kinase family have been cloned. Twoadditional subtypes have been discovered, p38 MAP kinase and c-junkinase (JNK) which may be activated independently and simultaneously.The ERK pathway is activated by growth factors or phorbol esters(Marshall, Cell 80:179-185, 1995). In contrast, p38 MAP kinase and JNKpathways are activated by inflammatory cytokines and cellular stressessuch as heat shock, osmotic stress or ultraviolet light(Galcheva-Gargova et al., Science 265:806-808, 1994; Kyriakis et al.,Nature 369:156-160, 1994; and Raingeaud et al., J. Biol. Chem.270:7420-7426, 1995).

Mammalian p38 MAP kinase was identified in murine pre-B cellstransfected with the LPS-complex receptor, CD 14, and in murinemacrophages where it is activated in response to LPS (Han et al.,Science 265:808-811, 1994). p38 has been identified as the mammalianhomologue of yeast osmosensing MAP kinase, HOG1 (Brewster et al.,Science 259:1760-1763, 1993), and the Xenopus kinase Mpk2 (Rouse et al.,Cell 78:1027-1037, 1994). CSBP1 and CSBP2 have been identified as humanhomologues of murine p38 MAP kinase (Lee et al., Nature 372:739-746,1994). p38 MAPK activation has also been identified in lymphocytesduring intra-thymic signaling essential for the differentiation andrepertoire selection of mature T-cell development (Sen et al., J.Immunol. 156:4535, 1996). Once activated by phosphorylation, p38 MAPKacts both transcriptionally and translationally to phosphorylatedownstream targets. Such targets include the transcription factorsATF-2, CHOP, HSP27, Max (Raingeaud et al., J. Biol. Chem. 270:7420,1995; Batchvarova et al., EMBO J. 14:4654, 1995; Freshney et al., Cell78:1039, 1994; and Zervos et al., Proc. Natl. Acad. Sci. USA 92:10531,1995), and the protein kinases MAPK activated protein kinase-2 and -3(MAPKAP-K2 and-K3) (McLaughlin et al., J. Biol. Chem. 271: 8488, 1996;and Beyaert et al., EMBO J. 15:1914, 1996).

p38 MAP kinase is activated in TNF-treated cells and plays a selectiverole in gene induction, controlling, for instance, synthesis of IL-6 andgranulocyte macrophage colony stimulating factor (GM-CSF) (Beyart etal., EMBO J. 15:1914-1923, 1996). The role of p38 in the signalingpathway for cytokine responses was further shown in studies using modelsystems of cytokine induction of HIV gene expression constructs tomeasure the effects of inhibiting p38. Using a laboratory cell linechronically infected with latent HIV, certain pharmaceutical inhibitorsof p38 MAPK were reported to block the cytokine-induced production ofHIV p24 (Shapiro et al., Eur. Cytokine Netw. 7:557, 1996). p38inhibitors were also reported to block the cytokine-specific inductionof HIV LTR-driven expression of an unrelated reporter molecule in atransfection model system (Kumar et al., Eur. Cytokine Netw. 7:558,1996).

In view of the multitude of roles played in intracellular signaling byp38 MAP kinase, there is a need in the art to find compounds that willinhibit intracellular signaling pathways through p38 MAP kinase. Suchselective inhibitors of this signaling pathway will inhibit T cellactivation and therefore show therapeutic utility for treatinginfections caused by retroviruses (for example, HIV) and for variousautoimmune diseases (for example, rheumatoid arthritis, lupus, graftversus host, host versus graft, insulin-dependent diabetes, and multiplesclerosis).

SUMMARY OF THE INVENTION

The present invention provides therapeutic modalities for the treatmentof diseases and disorders caused as a result of T cell activation, inparticular HIV-infection, by virtue of the p38 MAPK cellular signalingpathway as a target of intervention. The present invention specificallyprovides guanylhydrazone-substituted compounds to treat disorders anddiseases related to T cell activation and HIV-infection. The presentinvention also relates to the use of other compounds which inhibit thep38 MAP kinase pathway in treating immune diseases, and other diseasesor clinical conditions in which T lymphocytes and cytokines and/or othermediators released by activated T lymphocytes are implicated in thepathology of the disease condition.

The present invention also relates to therapeutic modalities for thetreatment of disorders and diseases related to T cell activation, inparticular HIV-infection, by administering an effective amount of a p38MAPK pathway inhibitor in combination with at least one othertherapeutic agent. Preferably the p38 MAPK pathway inhibitor is used incombination therapy with an antiviral agent of another class (i.e.,treatment mechanism of action), such as a reverse transcriptaseinhibitor (e.g., AZT, ddI, ddC, 3TC); an HIV protease inhibitor (e.g.,ABT-538); or a preintegration complex inhibitor.

The invention is based on the discovery that the p38 MAPK signaltransduction pathway participates in T lymphocyte co-stimulation. p38MAPK activity was found to be enhanced in human T lymphocytes stimulatedwith a-CD3 and a CD28. Inhibition of the p38 MAPK pathway, by theaddition of an exemplary multivalent guanylhydrazone, CNI-1493, resultedin inhibition of activation of the T lymphocytes, as measured by 1L-2synthesis.

The various modalities of treatment described herein were designed onthe basis of the proposed model. In particular, multivalentguanylhydrazone compounds, small organic molecules, antisense molecules,ribozymes and triple helix molecules targeted to the p38 MAP kinasepathway may be used to inhibit T cell activation and HIV-infection. In aspecific embodiment of the invention, multivalent guanylhydrazonecompounds, such as CNI-1493, may be used to inhibit HIV-infection. Inyet another embodiment of the invention, upstream and downstreamcomponents of the p38 MAP kinase pathway may be targeted to inhibitHIV-infection. For example, MAPKAP kinase-2, which is downstream of p38MAP kinase, may be targeted by antisense, ribozyme or triple helixmolecules, to inhibit HIV-infection.

The present invention further relates to screening assays to identifycompounds which inhibit the p38 MAP kinase pathway and may be used totreat diseases or disorders related to T cell activation, in particularHIV-infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the protein immunoblot result of phospho-p38 MAPK levelsin T cells in the absence (−) or presence (+) of HIV at various timepoints after infection.

FIG. 1B shows the cumulative results of similar experiments on threedonors (mean+/−standard error). These data show increasedphosphorylation of p38 MAPK after primary infection of T cells.

FIG. 2A shows that RAW 264.7 cells were pretreated with/without variousconcentrations of CNI-1493 one hour prior to addition of LPS(Escherichia coli 0111:B4 100 ng/ml; Sigma Chemical Co. St. Louis, Mo.).Cell lysates were prepared for immunoprecipitation and immunoblotting 15minutes after administration of LPS. In FIGS. 2B, 2C and 2DPHA-activated T cells were pretreated with control diluent or variousconcentrations of CNI-1493 for one hour, infected with HIV 1-LAV as inFIG. 1, and cultured for 10 days. FIG. 2B is a graph of dose-dependentinhibition by CNI-1493 of HIV-infection as assessed by the accumulationof reverse transcriptase (RT) at day 6 of culture. FIG. 2C is a graph ofHIV inhibition by 1 mM CNI-1493 over time. FIG. 2D is an immunoblot ofphospho-p38 MAPK expression in HIV-1 infected cells on day 6 afterculture treated with/without 1 mM CNI-1493. These data show inhibitionof HIV replication by the exemplary guanylhydrazone substituted compoundCNI-1493.

FIG. 3 shows that an 18-mer phosphothiorate oligonucleotides coding forbp 324-341 in either the sense (5′-GCAGGAGCTGAACAAGAC-3′ SEQ. ID. NO.:1)or antisense (5′-GTCTTGTTCAGCTCCTGC-3′ SEQ. ID. NO.:2) direction wereused based on the sequence for human p38 MAPK (Han et al., 1995 Biochem.Biophys. Acta 1265:224). T cells were infected with HIV1-LAV in thepresence of 1 mM 18-mer, refed every 3-4 days with media containingfresh oligomer and supernatants collected at various time points for RTassay. Simultaneously, cells were also plated, treated similarly andharvested for phospho-p38 MAPK levels assayed by immunoprecipitation atvarious time points as measured by RT assay. Shown in this figure is theeffect of addition of sense and antisense p38 MAPK oligonucleotide onphospho-p38 levels and its correlation with HIV replication, as measuredby RT. These data show that phosphothiorate antisense p38 MAPKoligonucleotides inhibit HIV replication in T cells.

FIG. 4 shows the results of an experiment wherein T cells, prepared andinfected with HIV1-LAV (as described above), were grown and supernatantsharvested and assayed for-MIP-1α and MIP-1 levels by sandwich ELISA(Schmidtmayerova et al., 1996, Proc. Natl. Acad. Sci. USA 93:700)).Assays were done in triplicate and error bars represent standarddeviation of the mean. The data shown here is the result of anindividual donor experiment, showing that chemokine secretion of T cellsis unchanged or slightly increased during CNI-1493-caused inhibition ofHIV replication.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

“p38 MAP kinase” refers to the related homolog or analog belonging tothe mammalian family of p38 kinase activity, including, for instance,the human members of the family, CSBP1 and CSBP2.

“p38 MAPK signaling pathway” or “p38 MAPK pathway” refers to both theupstream and downstream components of the signaling cascade.

Guanylhydrazone-Substituted Therapeutic Compounds

The present invention provides a method for treating retroviral-causedinfection and autoimnune diseases, comprising administering an effectiveamount of a multivalent guanylhydrazone compound. The genera ofguanylhydrazone compounds to be included in the present invention isdescribed herein. The synthesis of guanylhydrazone compounds is alsoprovided by the present invention and is also described in U.S. Pat. No.5,599,984, incorporated herein by reference in its entirety. The terms“GhyCH—” shall mean NH₂(CNH)—NH—N═CH— and “GhyCCH₃—” shall meanNH₂(CNH)—NH—N═CCH₃—. The guanylhydrazone-substituted compounds shallinclude formula 1:

wherein X₂=GhyCH—, GhyCCH₃— or H—; X₁, X′₁ and X′₂ independently=GhyCH—or GhyCCH₃—; Z=—NH(CO)NH—, —(C₆H₄)—, —(C₅NH₃)— or —A—(CH₂)_(n)—A—,n=2-10, which is unsubstituted, mono- or di-C-methyl substituted, or amono or di- unsaturated derivative thereof; and A,independently,=—NH(CO)—, —(CO)NH—, —NH(CO)NH—, —NH— or —O— and saltsthereof. For ease of synthesis, a preferred embodiment includes thosecompounds wherein A is a single functionality. Also included arecompounds having the same formula 1 wherein X₁ and X₂=H; X′₁ and X′₂independently=GhyCH— or GhyCCH₃—; Z=—A—(CH₂)_(n)—A—, n=3-8; andA=—NH(CO)—,—(CO)NH— or —NH(CO)NH—, and salts thereof. Also included arecompounds wherein X₁, and X₂=H; X′₁ and X′₂ independently=GhyCH— orGhyCCH₃— and Z=—O—(CH₂)₂—O—.

Further examples of genera include: The genus wherein: X₂=GhyCH—,GhyCCH₃— or H—; X₁, X′₁ and X′₂=GhyCH— or GhyCCH₃—; andZ=—O—(CH₂)_(n)—O—, n=2-10 and salts thereof; and the related genuswherein, when X₂ is other than H, X₂ is meta or para to X₁ and whereinX′₂ is meta or para to X′₁. A compound having the above formula wherein:X₂=GhyCH, GhyCCH₃ or H; X₁, X′₁ and X′₂,=GhyCH— or GhyCCH₃—; andZ=—NH—(C═O)—NH— and salts thereof; and the related genus wherein, whenX₂ is other than H, X₂ is meta or para to X₁ and wherein X′₂ is meta orpara to X′₁.

Also included are compounds having a formula 2:

wherein: n=3-8; X₂ and X′₂=GhyCH—, GhyCCH₃— or H—; X₁ and X′₁=GhyCH— orGhyCCH₃—; and salts thereof; and the related genus wherein, when X₂ orX′₂ or both are other than H, then X₂ or X′₂ are meta or para to X₁ orX′₁, respectively.

The compounds of the present invention can be synthesized by means oftwo fundamental reactions. Numerous variants may be synthesized by meansof these reactions and that these variants have properties in commonwith the compounds herein.

Reaction 1 consists of the reaction of a substituted aromatic having aprimary or secondary amine, e.g., 3,5-diacetylaniline, and a dioyldichloride, e.g., glutaryl dichloride, to yield the correspondingN,N′-diphenylalkanediamide. “Reversed” diamides can also be prepared.Acetyl and diacetylbenzoic acid can be prepared by the reaction of thecorresponding substituted toluenes and KMnO₄. The acids may be thenactivated by standard techniques and reacted with the appropriateα,ω-alkanediamines to yield the reverse “diamides”. Mixed forward andreversed diamides can be synthesized by methods well known in the fieldof peptide synthesis. Thus, an N-t-butyloxycarbonyl amino acid may bereacted with a substituted aniline, followed by deprotection andreaction of the amino group with an activated substituted benzoic acid.When used herein the symbol “—NH(CO)—”, unless otherwise indicated,includes the —(CO)NH— isomer.

The method is not limited to dioyl dichlorides. The trichloridederivatives of trioyl compounds may be used to synthesize triphenylalkanetriamides in a similar fashion. Suitable triacids include cyclicacids, e.g., 1,3,5-cyclohexanetricarboxylic acid (Aldrich Chem. Co.),1,3,5-trimethyl,1,3,5-cyclohexanetricarboxylic acid (Kemp's triacid,Kemp and Petrakis, 1981, J. Org. Chem. 46:5140),1,3,5-benzinetricarboxylic acid (Aldrich Chem. Co.) and lineartricarboxylic acids such as 1,2,3-propanetricarboxylic acid (Sigma Chem.Co.). The identical reaction may be performed wherein the dioyl chlorideis replaced by trichloromethyl chloroformate to yield a diphenylureacondensation product. An alternative to Reaction 1 can be performed toyield a 1,n-(n-alkanedioxy) diarylene by reacting the 1,n-dibromoalkane,e.g., 1,2 dibromoethane and a monohydroxylarylene, e.g.,3-hydroxyacetophenone.

Further embodiments of the invention include the use of triamines of theform H₂N—(CH₂)_(n)—NH—(CH₂)_(q)—NH₂ wherein (n,q=2-6) and of the formY—((CH₂)_(n)—NH₂)₃ wherein Y may be one of N (n=2-6), C(NO₂)(n=3), aC-alkane (n=1), 1,3,5-adamantanetriyl (n=3) or 1,3,5-benzinetriyl(n=1-3).

In two further embodiments, an acetyl- or diacetylaryl isocyanate isreacted with an alkanediamine or, alternatively, an acetyl- ordiacetylaryl amine is reacted with an alkanediyl diisocyanate to yieldbis-ureido intermediates which may be reacted with aminoguanidine toform the guanylhydrazone end products. The requisite isocyanates areeither commercially available or may be synthesized from thecorresponding amines by reaction with phosgene, trichloromethylchloroformate, or bis(trichloromethyl) carbonate in toluene or xylene atelevated temperature.

Reaction 2 consists of the reaction of an acetophenone or benzaldehydetype moiety and an aminoguanidine to yield the condensation productwherein an imino-bonded (N═C) aminoguanidine replaces the ketone orcarbonyl moiety of the arylene thus forming a guanylhydrazone andaccompanied by the release of a water molecule.

P38 MAP Kinase Pathway

The present invention relates to therapeutic modalities andpharmaceutical compositions which target the p38 MAP kinase pathway forthe treatment of diseases and disorders related to T cell activation, inparticular HIV-infection. The present invention relates to a variety oftechniques and compositions which may be utilized to inhibit T cellactivation and HIV-infection. Such techniques may include, but are notlimited to, gene therapy approaches, drugs, small organic moleculesidentified to inhibit the p38 MAP Kinase pathway.

Without being limited by any theory regarding the mode of action of themultivalent guanylhydrazones, these compounds may work by disrupting thep38 MAPK pathway. p38 MAP kinase pathway plays a critical role in theactivation of human T lymphocytes and this pathway is inhibited by themultivalent guanylhydrazone compounds. Moreover, treatment of T cellcultures with multivalent guanylhydrazone compounds inhibitedHIV-infection, as did inhibition of the p38 MAPK pathway by antisensemolecules directed to the p38 MAPK sequence. The p38 MAP kinase pathwayis activated when T lymphocytes are stimulated with either an antibodyto CD3 or an antibody to CD28, and significantly activated when Tlymphocytes were co-stimulated with both antibodies. The activation ofthe p38 MAP kinase pathway was inhibited by the addition of amultivalent guanylhydrazone to the stimulated T lymphocytes. Inaddition, the stimulation of the T lymphocytes was also inhibited by theaddition of the multivalent guanylhydrazone, as measured by IL-2secretion.

The necessity of p38 MAP kinase activation in HIV-infection of T cellswas determined from experiments in which p38 MAPK activity was blockedby the addition of specific inhibitors of p38 MAPK expression, p38 MAPKantisense oligonucleotides. The necessity of p38 MAPK pathway activationto support HIV-infection was further demonstrated by the ability of p38MAPK pathway inhibitor, CNI-1493, a multivalent guanylhydrazone, toinhibit HIV-infection in primary T cells. These results show that thep38 MAP kinase pathway plays a critical role in T cell activation and inthe ability of T cells to support HIV-infection.

Among the compounds useful to disrupt the activity of p38 MAP kinase andthe other components of its signaling pathway are antisense, ribozymeand triple helix molecules. Such molecules are designed to inhibitexpression of the target genes, p38 MAP kinase or the other componentsof its signaling pathway.

Anti-sense RNA and DNA molecules act to directly block the translationof mRNA by hybridizing to targeted mRNA and preventing proteintranslation. With respect to antisense DNA, oligodeoxyribonucleotidesderived from the translation initiation site, that is, between the −10and +10 regions of the target gene nucleotide sequence of interest, arepreferred.

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various well-known modifications to the DNA molecules may be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include but are not limited to, the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

Treatment of Autoimmune Diseases Characterized by T Cell Activation

The present invention relates to the use of compounds which inhibit Tcell activation and/or which inhibit the p38 MAP kinase pathway as amethod of treating disorders related to T cell activation. In particularthe invention relates to the use of multivalent guanylhydrazonecompounds and antisense molecules as a method of treating disordersrelated to T cell activation. The compounds of the present inventionhave utility in treating disorders which result from an inappropriate,rather than an insufficient immune response. Examples of such disorders,include but are not limited to, atopic conditions (IgE-mediated allergicconditions), such as asthma, allergy, including allergic rhinitis,dermatitis, including psoriasis, pathogen susceptibilities, chronicinflammatory disease, organ-specific autoimmunity including multiplesclerosis, Hashimoto's thyroiditis and Grave's disease, graft rejection,and graft-versus-host disease. Other immune disorders involving T cellactivation include, but are not limited to, chronic inflammatorydiseases and disorders, such as Crohn's disease, systemic lupuserythematosus, myasthenia gravis, thyroiditis, reactive arthritis,including Lyme disease, insulin-dependent diabetes, contact dermatitis,gastrointestinal allergies, including food allergies, eosinophilia,conjunctivitis, glomerular nephritis, certain pathogen susceptibilitiessuch as helminthic (leishmaniasis), gram positive superantigen-inducedshock, and certain viral infections, including HIV, and bacterialinfections, including tuberculosis and lepromatous leprosy.

As described above, because of their pharmaceutical properties, thecompounds of the present invention can be used especially as agents totreat patients suffering from disorders related to T cell activation.Such compounds can be administered to a patient either alone, or inpharmaceutical compositions where it is mixed with suitable carriers orexcipient(s).

Screening Assay

The following assays are designed to identify compounds that interferewith or inhibit the p38 MAP kinase signaling pathway and, as a result,inhibit the activation of T cells. Compounds may include, but are notlimited to, guanylhydrazone-substituted compounds according to formula 1or formula 2 herein, and anti-p38 MAPK antisense oligonucleotides. Thosecompounds identified as inhibitors of the p38 MAP kinase pathway wouldhave utility in treatment of disease or disorders related to T cellactivation.

The assay identifies compounds which inhibit p38 MAP kinase pathway and,as a result, T cell activation. The assays of the present invention mayinclude in vitro kinase assays which measure the effects of testcompounds on the individual components of the p38 MAP kinase signalingpathway. For example, but not by limitation, these assays may involvemeasuring the effects of a test compound on a reaction mixturecontaining the test compound and a cell lysate prepared from activated Tcells and a sufficient amount of time for the components to interact.p38 MAP kinase or another component of the pathway, such as MAPKAP-K2,is immunoprecipitated from the cell lysate and its kinase activitymeasured using a known substrate of the kinase, i.e., transcriptionfactors AFT-2, CHOP, HSP27 and Max. The activity of the p38 MAP kinasecomponent will be determined as a measurement of the phosphorylatedstate of the substrate. In order to test a compound for inhibitoryactivity, the reaction mixture is prepared in the presence and absenceof the test compound. Control reaction mixtures are incubated withoutthe test compound or with a placebo. The lack of phosphorylation of thesubstrate indicates that the MAP kinase is inhibited from interactingwith or phosphorylating its substrate.

In a particular embodiment, the target substrate can be prepared forimmobilization using recombinant DNA techniques routinely used in theart. For example, the target gene coding region can be fused to aglutathione-S-transferase (GST) gene using a fusion vector, such aspGEX-5X- 1, in such a manner that its conformation is maintained in theresulting fusion protein. In such an assay, the GST-target gene fusionprotein can be anchored to glutathione-agarose beads. The activated Tcell lysate can be added in the presence or absence of the test compoundin a manner that allows the kinase reaction to occur in the presence of³²P-ATP. At the end of the reaction period, unbound material can bewashed away, and the substrate assayed for its phosphorylated state. Theinteraction between the target gene protein and the p38 MAP kinasepathway component can be detected by measuring the amount ofradioactivity that remains associated with the glutathione-agarosebeads. A successful inhibition of the interaction by the test compoundwill result in a decrease in measured radioactivity.

Alternatively, the GST-target gene fusion protein and the interactivecellular extract can be mixed together in a kinase buffer in thepresence of ³²P-ATP, but in the absence of the solid glutathione-agarosebeads. The test compound can be added either during or after the speciesare allowed to interact. This mixture can then be added to theglutathione-agarose beads and unbound material is washed away. Again theextent of inhibition of the kinase reaction between the substrate targetgene product and the component of the p38 MAP kinase pathway can bedetected by measuring the radioactivity associated with the beads.

In addition, the assays of the present invention may include other invivo assays to measure the effects of the test compounds on T cellactivation. For example, test compounds may be assayed for their abilityto inhibit activation of T cells. In such assays, test compounds may beadded to T cells co-stimulated with a-CD3 and a-CD28. The stimulation ofthe T cells should result in the synthesis and secretion of cytokines,such as IL-2. The inhibition of the secretion or synthesis of suchcytokines indicates that the test compound inhibits the activation of Tcells.

Anti-Retroviral Combination Therapy

According to the present invention, multivalent guanylhydrazonecompounds and inhibitors of the p38 MAPK pathway may be used incombination therapy for the treatment of retroviral infections, inparticular HIV-infection. The guanyhydrazone-substituted compounds areused in combination with another antiviral agent or multiple antiviralagents to improve the overall antiviral effect. Such additionalantiviral agents which may be used with multivalent guanylhydrazonecompounds and p38 MAPK pathway inhibitors include but are not limited tothose which function on a different target molecule involved in viralreplication, such as, reverse transcriptase inhibitors, viral proteaseinhibitors, preintegration complex inhibitors, and glycosylationinhibitors.

Inhibitors of p38 MAPK pathway or a pharmaceutically acceptablederivative thereof can also be used in combination with retrovirusinhibitors, such as reverse transcriptase inhibitors, HIV proteaseinhibitors and preintegration complex inhibitors. Reverse transcriptaseinhibitors include 3′azido-3′-thymidine (AZT) and dideoxyinosine (ddI),2′,3′-dideoxyadenosine (ddA); 2′,3′-dideoxyguanosine (ddG);2′,3′-dideoxyinosine (ddI); 2′,3′-dideoxycytidine (ddC);2′,3′-dideoxythymidine (ddT); 2′,3′-dideoxy-dideoxythymidine (d4T) and3TC (sold under the brand name of Epivir®);2′,3′-dideoxy-2′-fluoronucleosides; 2′,3′-dideoxy-2′-fluoroadenosine;2′,3-dideoxy-2′-fluoroinosine; 2′,3′-dideoxy-2′-fluorothymidine;2′,3′-dideoxy-2′-fluorocytosine; and2′,3′-dideoxy-2′,3′-didehydro-2′-fluoronucleosides; and2′,3′-dideoxy-2′,3′-didehydro-2′-fluorothymidine (Fd4T). Preferably, the2′,3′-dideoxy-2′-fluoronucleosides of the invention are those in whichthe fluorine linkage is in the beta configuration, including, but notlimited to, 2′3′-dideoxy-2′-beta-fluoroadenosine (F-ddA),2′,3′-dideoxy-2′-beta-fluoroinosine (F-ddI), and2′,3′-dideoxy-2′-beta-fluorocytosine (F-ddC). Such combinations allowone to use a lower dose of the nucleoside derivative thus reducing thetoxicity associated with that agent, without loss of antiviral activitybecause of the use of the p38 MAPK pathway inhibitor. Moreover, such acombination reduces or avoids viral resistance, as the host cell ratherthan the virus is the target of the p38 MAPK inhibitors of the presentinvention.

Preferred combinations of p38 MAP kinase pathway inhibitors andnucleoside derivatives within the scope of the present invention includean effective amount of a multivalent guanylhydrazone-substitutedcompound and an effective amount of AZT to treat HIV-infection; and aneffective amount of a multivalent guanylhydrazone compound and aneffective amount of ddI, or an effective amount of a preintegrationcomplex inhibitor, such as those described in U.S. Pat. No. 5,574,040,the disclosure of which is incorporated by reference herein.

Inhibitors of the p38 MAP kinase pathway can also be used in combinationwith uridine phosphorylase inhibitors, including acyclouridinecompounds, including benzylacyclouridine (BAU);benzyloxybenzylacyclouridine (BBAU); aminomethyl-benzylacyclouridine(AMBAU); aminomethyl-benzyloxybenzylacyclouridine (AMB-BAU);hydroxymethyl-benzylacyclouridine (HMBAU); andhydroxymethyl-benzyloxybenzylacyclouridine (HMBBAU).

According to the present invention, inhibitors of p38 MAPK pathway or apharmaceutically acceptable derivative thereof can also be used incombination with cytokines or cytokine inhibitors, including but notlimited to rIFN α, rIFN β, rIFN γ, IL-2, inhibitors of TNFα, andMNX-160. Human rIFN-αA (>108 IU/mg) and rIFN γ(1.4×108 IU/mg) can beobtained from Hoffman LaRoche. Human rIFN β Ser 17 (1.0×108 IU/mg) areobtained from Berlex Biosciences. IL-2 (interleukin-2) can be obtainedcommercially from Chiron. Reference standards are obtained from theWorld Health Organization (human IFNα WHO standard B,69,19 and human IFNβ, WHO no. G-023-902-527, or the National Institute of Allergy andInfectious Disease, National Institute of Health no. G-023-901-530.

Inhibitors of p38 MAPK pathway can be used in combination with viralprotease inhibitors, including but not limited to, MK-639 (Merck),Invirase (saquinavir, Roche), ABT-538 (Abbott, CAS Reg. No.155213-67-5), AG1343, VX0478 (Burroughs Wellcome/Glaxo, CAS Reg. No.161814-49-9), DMP450, SC-52151 (Telinavir). Protease inhibitors aregenerally thought to work primarily during or after assembly (i.e.,viral budding) to inhibit maturation of virons to a mature infectiousstate. For example, ABT-538 has been shown to have potent antiviralactivity in vitro and favorable pharmokinetic and safety profiles invivo (Ho et al., Nature 373:123-126, 1995). Administration of ABT-538 toAIDS patients causes plasma HIV-1 levels to decrease exponentially andCD4 lymphocyte counts to rise substantially. The exponential decline inplasma viraemia following ABT-538 treatment reflects both the clearanceof free virions and the loss of HIV-1 producing cells as the drugsubstantially blocks new rounds of infection. ABT-538 treatment reducesvirus-mediated destruction of CD4 lymphocytes. Combining this treatmentwith inhibitors of the p38 MAP kinase pathway, which inhibits at adifferent stage of HIV-infection, would be likely to have synergisticeffects and have a dramatic clinical impact.

Inhibitors of p38 MAPK pathway or a pharmaceutically acceptablederivative thereof can also be used in combination with a class ofanti-HIV drugs which interfere with 5′-mRNA processing, for exampleribavirin. (Ribavirin (Virazole) from Viratel Inc.). Although themechanism of action of ribavirin is not clear, this drug is thought tocompete with guanosine in the formation of mRNA cap structures and/orinterfere with the functional methylation of these molecules. Inaddition, inhibitors of p38 MAPK pathway can be used in combination withtherapeutic agents, such as Amphotericin B (Fungizone, obtained fromGibco) a polyene microlide antifungal antibiotic which interacts withsterols and binds to them irreversibly. Amphotericin B represents aunique class of agents that are active against a variety of lipid-enveloped viruses, including HIV. Although amphotericin B exhibitssevere in vivo toxicities, the methyl ester form of this drug alsoexhibits anti-HIV activity and has a low cellular toxicity profile invitro. Therefore amphotericin B or its methyl ester can be used incombination therapy with inhibitors of p38 MAPK pathway.

Inhibitors of the p38 MAPK pathway can also be used in combination withinhibitors of glycoprotein processing, such as castonospermine(Boehringer Mannheim). Castanospermine is a plant alkaloid whichinhibits glycoprotein processing, and acts as an anti-HIV since HIVcontains two heavily glycosylated proteins, gp120 and gp41. Proteinglycosylation plays an important role in gp 120 interaction with CD4.Under conditions of infection by progeny virions synthesized in thepresence of castanospermine, the infectivity of HIV was attenuated.

Preferred combinations to be used within the methods of treating HIVinclude the use of an effective amount of a multivalentguanylhydrazone-substituted compound and an effective amount of ddI; theuse of an effective amount of a multivalent guanylhydrazone compound andan effective amount of 3TC; and the use of an effective amount of amultivalent guanylhydrazone-substituted compound and an effective amountribavirin.

Therapeutic Uses, Routes of Administration and Formulations

The inhibitors of p38 MAPK pathway may be used for antivirals effectsagainst viral infection other than HIV, such as HBV, EBV, CMV, and otheropportunistic infections including TB. Effective doses of thecombination therapy as described below may be formulated in suitablepharmacological carriers and may be administered by any appropriatemeans, including, but not limited to injection (intravenous,intraperitoneal, intramuscular, subcutaneous), by absorption throughepithelial or mucocutaneous linings (oral mucosa, rectal and vaginalepithelial linings, nasopharyngeal mucosa, intestinal mucosa); orally,transdermally or any other means available within the pharmaceuticalarts.

A compound can be administered to a human patient by itself or inpharmaceutical compositions where it is mixed with suitable carriers orexcipients at doses to treat or ameliorate various conditions involvingT cell activation and viral infection. A therapeutically effective dosefurther refers to that amount of the compound sufficient to inhibit Tcell activation and/or HIV-infection. Therapeutically effective dosesmay be administered alone or as adjunctive therapy in combination withother treatments for HIV-infection or associated diseases. Techniquesfor the formulation and administration of the compounds of the instantapplication may be found in “Remington's Pharmaceutical Sciences” MackPublishing Co., Easton, Pa., latest addition.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, and optionallyin a depot or sustained release formulation.

Furthermore, one may administer the agent of the present invention in atargeted drug delivery system, for example in a liposome coated with ananti-CD4 antibody. The liposomes will be targeted to and taken upselectively by cells expressing CD4.

The pharmaceutical compositions may be manufactured by means ofconventional mixing, dissolving, dragee-making, levitating, emulsifying,encapsulating, entrapping, or lyophilizing processes. Pharmaceuticalcompositions for use in accordance with the invention may be formulatedin conventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers, such asHank's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are usually knownin the art.

For oral administration, the compounds can be formulated by combiningthe active compounds with pharmaceutically acceptable carriers. Suchcarriers enable the compounds to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated. Pharmaceuticalpreparations for oral use can be obtained solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner. For administration byinhalation, the compounds for use according to the invention aredelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, such as,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, such as, by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulary agents such assuspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle before injection, such as, sterilepyrogen-free water.

Liposomes and emulsions are known examples of delivery vehicles orcarriers for hydrophobic drugs. Certain organic solvents such asdimethylsulfoxide also may be employed, although usually at the cost ofgreater toxicity. Additionally, the compounds may be delivered using asustained-release system, such as semipermeable matrices of solidhydrophobic polymers containing the therapeutic agent. Various ofsustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the therapeutic reagent, additional strategies for proteinstabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Many of the compounds of the invention identified as inhibitors of thep38 MAPK signaling pathway may be provided as salts withpharmaceutically compatible counterions. Pharmaceutically compatiblesalts may be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.;or bases. Salts tend to be more soluble in aqueous or other protonicsolvents that are the corresponding free base forms. Examples ofpharmaceutically acceptable salts, carriers or excipients are well knownto those skilled in the art and can be found, for example, inRemington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, Ed.,Mack Publishing Co., Easton, Pa., 1990. Such salts include, but are notlimited to, sodium, potassium, lithium, calcium, magnesium, iron, zinc,hydrochloride, hydrobromide, hydroiodide, acetate, citrate, tartrate,malate salts, and the like.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve their intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. Such information can be used to more accuratelydetermine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in a reduction in the intensity of the infection or inamelioration of symptoms or a prolongation of survival in a patient.Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical, pharmacological, and toxicological proceduresin cell cultures or experimental animals for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio between LD₅₀ and ED₅₀. Compounds whichexhibit high therapeutic indices are preferred. The data obtained fromcell culture assays or animal studies can be used in formulating a rangeof dosage for use in humans. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ (with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain thedesired modulating effects, or minimal effective concentration (MEC).The MEC will vary for each compound but can be estimated from in vitrodata; such as, the concentration necessary to achieve a 50-90%inhibition of HIV-infection using the assays described herein. Dosagesnecessary to achieve the MEC will depend on individual characteristicsand route of administration. However, HPLC assays, bioassays orimmunoassays can be used to determine plasma concentrations.

The amount of composition administered will be dependent on the subjectbeing treated, on the subject's weight, the severity of the affliction,the manner of administration and the judgment of the prescribingphysician.

EXAMPLE 1

This example illustrates an experiment showing that an exemplaryguanylhydrazone-substituted compound, CNI-1493(N,N′-bis(3,5-diacetylphenyl)decanediamide tetrakis(amidinohydrazone)tetrahydrochloride )inhibited the p38 MAP kinase pathway. CNI-1493 hasbeen shown to inhibit TNF production by macrophages activated by LPS.CNI-1493 blocked the synthesis of a 26 kDA membrane form of TNF,indicating that protein translation was inhibited. Further evidence forthe mechanism of translation suppression was given by experiments usingconstructs in which the 5′UTR and the 3′UTR of TNF to drive expressionof a reporter molecule. Both the 5′UTR and the 3′UTR regions of the TNFgene were required to elicit maximal translation suppression byCNI-1493. The effect of CNI-1493 on p38 MAPK was next determined as p38MAPK has been implicated in the regulation of TNF translation. Thepretreatment of human PBMC's with CNI-1493, resulted in the suppressionof p38 MAPK activity after the addition of LPS. These results indicatesthat CNI- 1493 specifically inhibits activation of the p38 MAPK signaltransduction pathway in LPS-mediated TNF production.

EXAMPLE 2

This example illustrates an experiment showing that the p38 MAP kinasepathway participates in T lymphocyte co-stimulation. Co-stimulation of Tlymphocytes occurs when two independent signals are applied to T cells,such as immobilized α-CD3 and soluble α-CD28, resulting in T cellactivation and, among other manifestations, production of IL-2. Twomembers of the MAPK family of signal transduction molecules, ERK andJNK, have previously been implicated in T cell co-stimulation. p38 MAPKhas been shown to play a role in during the activation of monocytes andmacrophages, however little is known about the role p38 MAPK function inT cells.

For studies of human T cells, buffy coats were obtained by elutriationfrom normal human donors to the Long Island Blood Bank Services.Peripheral blood mononuclear cells (PBMC's) were isolated by densitygradient centrifugation through Ficoll-Hypaque (Hypaque, Pharmacia);typically one preparation yielded 200×10⁶ cells. T cells were isolatedby passing through a T cell column (R&D) as per manufacturer'sinstructions. Jurkat cells were obtained from ATCC and maintained inDMEM containing 10% heat-inactivated fetal bovine serum, antibiotics andL-glutamine.

A rabbit polyclonal antibody against p38, and the recombinant GST-AFT2plasmid where a gift of Dr. Roger Davis (Howard Hughes Institute,Worcester, Mass.). A GST-AFT2 fusion protein was purified by affinitychromatography using glutatione-agarose, as per manufacturer'sinstructions (Pharmacia, Inc.). The human monoclonal anti-CD3 antibodywas purchased from Pharmingen and a human monoclonal anti-CD28 antibodywas purchased.

Whole cell lysates were prepared using kinase lysis buffer, containing afinal concentration of 20 mM Tris pH 7.5, 10% glycerol, 1% Triton X-100,0.137 M NaCl, 25 mM B-glycerophosphate, 2 mM EDTA, 1 mM orthovanadate, 2mM pyrophosphate, 10 μg/ml Leupeptin, 1 mM PMSF. Proteins werefractionated on a 12% SDS-PAGE gel, transferred by electrophoresis toPVDF membranes (Biorad, Inc.) and probed with a 1:1000 dilution of arabbit polyclonal antibody that immunospecifically recognizesphosphorylated human p38 MAPK (Santa Cruz Biotechnology, Inc.). Bindingto antibody was detected using enhanced chemiluminescence (AmershamInternational PLC).

2-5×10⁶ cells were lysed after appropriate pretreatment in 500-1000 μlkinase lysis buffer and centrifuged at 14,000×g for 15 minutes at 4° C.Endogenous p38 was immunoprecipitated with polyclonal p38 antibodyprebound to protein-A agarose for one hour at room temp. Agarose beadswith immunoprecipitated p38 MAPK protein were washed twice with lysisbuffer described above, and kinase assays performed by washing twicewith kinase buffer (25 mM Hepes pH 7.4, 25 mM B-glycerophosphate, 25 mMMgCl₂ 2 mM dithiothreitol, 0.1 mM orthovanadate), and incubation with 5μg of substrate protein (GST-ATF2), and 50 μM (gamma-³²P) ATP (10Ci/mmol) in a final volume of 30 μl. The reactions were stopped after 30minutes by boiling in an equivalent volume of 2× Laemmli sample buffer.The phosphorylated substrate was visualized using SDS-PAGE byautoradiography and phosphorlmager analysis (Amersham, Inc.).

T cell co-stimulation resulted in synergistic p38 kinase activity. Totest whether p38 MAP kinase was stimulated during co-stimulation of Tcells, antibodies to CD3 and CD28 were used singly and in combinationagainst Jurkat cells in vitro. Jurkat cells were treated with eitherimmobilized anti-CD3, soluble CD28 or both for one half hour, harvestedand assayed for p38 MAP kinase activity by measuring the phosphorylationof a known p38 MAPK target, the transcription factor ATF2. Addition ofCD3 alone stimulated p38 MAPK activity 3 fold, whereas addition ofanti-CD28 alone stimulated p38 MAPK activity 5-fold. Co-stimulation byaddition of both anti-CD3 and anti-CD28 antibodies resulted insynergistic p38 MAPK activity, increasing 12-fold. Co-stimulation byaddition of both anti-CD3 and anti-CD antibodies resulted in synergisticp38 MAPK activity, increasing 12-fold over baseline levels, as measureddensitometrically.

p38 MAPK activation was inhibited by CNI-1493 in monocytes andmacrophages after stimulation with lipoplysaccharide, although it is notclear whether p38 MAPK or its upstream activators are the direct drugtarget. CNI-1493 was added to Jurkat cells exposed to anti-CD3,anti-CD28 or both anti-CD3+ anti-CD28 to test whether this agent couldinhibit the p38 MAPK activity induced by co-stimulation. The observedinhibition of p38 MAP kinase activity by CNI-1493 was dose-dependent.Although anti-CD3 stimulation was unaffected by CNI-1493, stimulation ofp38 MAPK was inhibited approximately 50%, and co-stimulation withanti-CD3 and anti-28 was inhibited completely at a dose of 1.5 μManti-CD28. Thus, CNI-1493 inhibited p38 MAP kinase activity stimulatedduring T cell co-activation.

EXAMPLE 3

This example illustrates a role for the p38 MAP kinase pathway in HIV-1infection of primary T cells, specifically addressing the hypothesisthat activation of p38 MAPK is required for HIV-1 infection of primary Tlymphocytes. For studies of human T cells, buffy coats were obtained byelutriation from normal human donors to the Long Island Blood BankServices. Peripheral blood mononuclear cells (PBMC's) were isolated bydensity gradient centrifugation through Ficoll-Hypaque (Hypaque,Pharmacia). Typically, one preparation yielded about 200×10⁶ cells. Tcells were isolated by passing through a T cell column (R&D) as permanufacturer's instructions. Jurkat cells were obtained from ATCC andmaintained in DMEM containing 10% heat-inactivated fetal bovine serum,antibiotics and L-glutamine.

T cells were prepared by Ficoll-Hypaque separation, adherence exclusionof monocytes, and activation with PHA (5 μg/ml) for 3 days in RPMI1640+10% FCS+L-glutamine+antibiotics (PS). After HIV 1-LAV stocks wereprepared and titrated on CEM cells, T cells were washed and incubatedwith 1000 tissue culture infectious units (TCIU) of virus for 2 hours at37 degrees C, washed and fresh medium added containing IL-2 20 U/ml. Atvarious time points after infection, cell lysates were prepared.

A rabbit polyclonal antibody against p38, and a recombinant GST-AFT2plasmid where a gift of Dr. Roger Davis (Howard Hughes Institute,Worcester, Mass.). A GST-AFT2 fusion protein was purified by affinitychromatography using glutatione-agarose as per manufacturer'sinstructions (Pharmacia, Inc.). Human monoclonal anti-CD3 antibody waspurchased from Pharmingen and the human monoclonal anti-CD28 antibodywas purchased.

Whole cell lysates were prepared using kinase lysis buffer containing afinal concentration of 20 mM Tris pH 7.5, 10% glycerol, 1% Triton X-100,0.137M NaCl, 25 mM B-glycerophosphate, 2 mM EDTA, 1 mM orthovanadate, 2mM pyrophosphate, 10 μg/ml Leupeptin, 1 mM PMSF. Proteins werefractionated on a 12% SDS-PAGE gel, transferred by electrophoresis toPVDF membranes (Biorad, Inc.) and probed with a 1:1000 dilution of arabbit polyclonal antibody that immunospecifically recognizesphosphorylated human p38 MAPK (Santa Cruz Biotechnology, Inc.). Bindingto antibody was detected using enhanced chemiluminescence (AmershamInternational PLC).

2-5×10⁶ cells were lysed after appropriate pretreatment in 500-1000 μlkinase lysis buffer and centrifuged at 14,000×g for 15 minutes at 4 ° C.Endogenous p38 was immunoprecipitated with polyclonal p38 antibodyprebound to protein-A agarose for one hour at room temp. Agarose beadswith immunoprecipitated p38 MAPK protein were washed twice with lysisbuffer described above, and kinase assays performed by washing twicewith kinase buffer (25 mM Hepes pH 7.4, 25 mM B-glycerophosphate, 25 mMMgCl₂ 2 mM dithiothreitol, 0.1 mM orthovanadate), and incubation with 5μg of substrate protein (GST-ATF2), and 50 μM (gamma-³²P) ATP (10Ci/mmol) in a final volume of 30 ill. The reactions were stopped after30 minutes by boiling in an equivalent volume of 2× Laemmli samplebuffer. The phosphorylated substrate was visualized using SDS-PAGE byautoradiography and phosphorimager analysis (Amersham, Inc.).

For anti-sense experiments, 18-mer phosphothiorate oligonucleotidescoding for bp 324-341 in either the sense (5′GCAGGAGCTGAACAAGAC3′ SEQ IDNO.: 1) or antisense (5′GTCTTGTTCAGCTCCTGC3′ SEQ ID NO.:2) directionswere used, based upon the sequence for human p38 MAPK (Han et al.,Biochem Biophys. Acta 1265:224, 1995). T cells were infected withHIV1-LAV in the presence of 1 μM 18-mer, refed every 3-4 days with mediacontaining fresh oligomer and supernatants collected at various timepoints for RT assay. Simultaneously, cells were also plated, treatedsimilarly and harvested for phospho-p38 MAPK levels assayed byimmunoprecipitation at varying time points. FIGS. 1-3 show the effect ofaddition of sense and antisense p38 MAPK oligonucleotide on phospho-p38levels and its correlation with HIV replication, as measured by RT.

Primary T lymphocytes were isolated from normal donors stimulated withPHA for 2-3 days, and then infected with the LAV strain of HIV. As earlyas 30 minutes post-infection, a rapid increase in the phosphorylation ofp38 MAP kinase was observed in HIV-infected cells, as compared tocontrol uninfected cells (FIGS. 1A and 1B). Levels of p38 MAP kinaseactivity remained elevated (450% increase as compared to uninfectedcells; p<0.05 for up to 2 hours following introduction of virus into thecultured T cells. The magnitude of p38 activation in T cells as aconsequence of HIV-infection correlated well with increases in p38 MAPKobserved in macrophages following activation by known stimuli of p38MAPK such as UV, TNF and LPS (Raingeaud et al., J. Biol. Chem.270:27395, 1995). The kinetics of p38 MAPK activation in the two celltypes were similar as well, remaining elevated for up to two hours butreturning to basal levels within 6 hours.

These results suggest the possibility that HIV-1 infection of T cellsrequires activation of the p38 MAPK pathway. Primary T cells activatedwith PHA were pretreated (or not) with CNI-1493 for one hour, infectedwith the LAV strain of HIV, and RT activity measured at various timepoints after infection, up to 10 days. As shown in FIG. 2B, CNI-1493inhibited HIV replication in T-cells in a dose-dependent manner, with anIC₅₀ of approximately 0.5 μM. A time course of the effect of CNI-1493(1μM) on HIV replication is shown in FIG. 2B. The effect of CNI-1493 onsuppression of p38 MAP kinase activity during this period correlatedwith its suppressive effect on HIV replication (FIGS. 2C, 2D).

Further evidence for the necessity of p38 MAPK activation in T cellHIV-1 infection was obtained from experiments in which p38 MAP kinaseactivity was blocked by the addition of antisense oligonucleotides.Phosphothiorated sense and antisense p38 MAPK oligomers were added toLAV-infected primary T cells simultaneously with the virus, and thenreplenished every three days thereafter until day 10. The p38 MAP kinaseantisense oligonucleotides decreased HIV replication proportionate tothe decrease in phospho-p38 MAPK activity on both day 7 and day 10 afterinfection (FIG. 3). Thus, two independent methods that suppress p38 MAPKactivation (CNI-1493 and antisense oligonucleotides) each inhibited HIVreplication, suggesting that activating p38 MAPK is required for maximalHIV replication.

The CC-chemokines MIP-1α, MIP-1β and RANTES have recently been shown toinhibit HIV replication in T cells (Geng et al., J. Immunol. 151:6692,1993) and it has been suggested these molecules may play a role in thehost defense against viral infection. Because ideal anti-HIV therapieswould not interfere with the ability of the host to mount its owndefense, it was also evaluated whether inhibition of the p38 MAP kinasesignal transduction pathway inhibits chemokine production in T cells.Levels of MIP 1α and MIP 1β during LAV infection of primary T cells werenot decreased by CNI-1493 (FIG. 4). MIP 1α and 1β levels droppedslightly late in HIV-1 infection (day 9). In the presence of CNI-1493,however, chemokine levels either were slightly elevated (day 6) ornormal (day 9). Thus, suppression of p38 MAP kinase activity duringHIV-infection of T cells did not adversely affect one mechanismcontributing to the cells' natural immunity against the virus, in theform of chemokine expression, while simultaneously inhibiting HIVreplication.

When considered together, these results identify the p38 MAP kinasesignal transduction pathway as critical for maximal in vitro infectivityof primary T-cells by HIV-1. Stimulation of p38 MAPK activity was early(within 30 minutes) after introduction of virus, and remained elevatedup to 9 days post-infection. Inhibition of the p38 MAPK pathway byCNI-1493, as well as by p38 antisense oligonucleotides was able toreduce HIV replication, as measured by RT. The secretion of chemokines,potent natural immunoinhibitory molecules against HIV, is unaffected.Hence, p38 MAPK kinase represents a novel therapeutic target againstprimary HIV-infection of T cells.

2 18 nucleic acid single unknown MAPKK sense 1 GCAGGAGCTG AACAAGAC 18 17nucleic acid single unknown MAPKK antisense 2 TCTTGTTCAG CTCCTGC 17

We claim:
 1. A method of treating a disorder or disease characterized byT cell activation, comprising administering to a subject in need thereofan effective amount of an agent, other than an inhibitor of kinaseactivity of the p38 MAPK, that inhibits gene expression of a componentof the p38 MAPK signaling pathway.
 2. The method of claim 1, wherein theagent is an antisense molecule that hybridizes to p38 MAPK mRNA.
 3. Amethod of treating a disorder or disease characterized by T cellactivation, comprising administering to a subject in need thereof anagent, other than an inhibitor of kinase activity of the p38 MAPK, thatinhibits expression of the p38 MAPK gene.